The Candidate: My long-term goal is to establish myself as an independent investigator in laboratory based research focused on the mechanisms of autoimmune diseases caused by dysregulated signal transduction in immune cells. I am seeking a KO1 Award in order to complete my training at UCSF and transition to a tenure- track position. My graduate training under the mentorship of Mark Schlissel was highly productive, resulting in five publications including two first-author articles and three second-author articles As I joined Dr. Weiss' laboratory as a postdoctoral scholar, I initiated a project aimed at definin the in vivo role of tyrosine 315 and 319 of ZAP-70 in regulating ZAP-70 function by generating a ZAP-70 knock-in mouse, the YYAA mouse. Interestingly, characterization of YYAA mice has led me to a new research interest --autoimmunity and immune tolerance. By comparing two ZAP-70 mutants in the SKG and YYAA mice, I have defined a threshold for autoimmunity versus autoimmune arthritis. These findings were published in Journal of Experimental Medicine in 2009. In a recent collaboration with my co-worker from Dr. Weiss lab, Byron Au-Yeung, we uncovered a previously overlooked kinase-independent function of ZAP-70 in the T regulatory cells. This collaborative work was published last year in Nature Immunology after my initial submission. My immediate goals for the duration of the award are 1) to acquire the necessary skills to identify pathogenic autoantigens and the T cells that recognize them; 2) to obtain guidance for maturation as a scientist and my preparation for an academic job search as well as to acquire training in management skills needed to lead a research group; 3) to develop experimental reagents that are critical in my goal of submitting a competitive RO1 application during my first two years as an independent investigator. I have assembled a mentoring committee that, in addition to my mentor Dr. Weiss, includes Drs. Mark Anderson, Abul Abbas and Mehrdad Matloubian. Each person brings a different level of expertise that is directly relevant to my research plan and, combined with my didactic training plan, will help me achieve my goals and successfully transition to an independent academic investigator. Environment: I believe UCSF provides the rich environment in studying autoimmunity. Using the expertise in the Weiss lab, I will gain extensive hands-on experience in biochemistry which is essential for the identification of tissue-specific autoantigens. Additionally, Dr. Weiss' lab is located in clos proximity to other faculty members at UCSF working on autoimmunity such as Dr. Abbas, and Dr. Anderson. Because of the close proximity, the collaborations and discussions are frequent and easily established. Although I will conduct my experiments mainly in the Weiss lab, I will gain expertise in trafficking of autoreactive T cells as well as autoimmunity/immune tolerance through the laboratories of Drs. Matloubian, Abbas, and Anderson. Abstract of Research Project: Rheumatoid arthritis (RA), one of the most common human autoimmune diseases, primarily affects the synovial membranes of joints. CD4+ T cells are thought to play an essential role in the development of RA, where they are a predominant cell type found in the inflamed joint. Despite intensive investigation, the initial phase of this disease remains uncharacterized, largely due to varied intervals before the onset of symptoms in patients. Mouse models of RA such as the SKG mouse, which shares many features with human RA, have become instrumental to study early events in the disease. Dr. Hsu's research seeks to define the cause and pathogenic mechanisms of RA using mice possessing hypormorphic mutants of ZAP-70, the SKG and YYAA mice. Dr. Hsu has previously demonstrated that both YYAA and SKG mice have impaired T cell development, hyporesponsiveness to TCR stimulation, and defective negative and positive selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Her findings suggested that the difference in TCR repertoires may be responsible for their differential susceptibility to arthritis. The central hypothesis of the proposed experiments is that distinct TCR repertoires selected in the YYAA and SKG mice determine a threshold for pathogenic autoreactive T cells leading to arthritis. The specific aims testing this hypothesis are as follows: (AIM1) understand the properties and kinetics of the accumulation of autoreactive T cells into the inflamed joints, (AIM2) identify arthritogenic T cells and a dominant autoantigen in SKG mice. In Aim 1, IFN3 and IL-17 reporter mice crossed to either YYAA or SKG mice will be used to define the roles of IFN3 and IL-17-producing cells in the initiation phase of autoimmune arthritis. BrdU pulse labeling experiments will be used to characterize the kinetics and migration patterns of activated autoreactive T cells in YYAA and SKG mice. In Aim 2, experiments will be conducted to compare YYAA and SKG mouse TCR repertoires of the infiltrating T cells in the joints and peripheral T cells from lymph nodes. Retroviral infection of a TCR1 library generated from purified infiltrating T cells in the inflamed joints will be combined with adoptive transfer experiments to identify and clone the arthritogenic T cells from SKG mice. These experiments will contribute to the fundamental understanding of the cellular mechanisms of RA which may provide useful targets for intervention. PUBLIC HEALTH RELEVANCE: With the increasing prevalence of rheumatoid arthritis and need for more effective targeted therapies, a better understanding of pathogenesis is required, in particular defining underlying mechanisms that contributes to the development of the disease. With this project, I hope to identify and characterize the pathogenic T cells as well as autoantigen leading to the development of autoimmune arthritis. I have two powerful mouse models that can be used to mechanistically address these questions and potentially identify new therapeutic targets.